This invention relates to a method of synthesizing within a bacterial host, and secreting through the membrane of the host, a selected protein or polypeptide, such as a eukaryotic cell protein, e.g., proinsulin, serum albumin, human growth hormone, parathyroid hormone and interferon. This invention particularly relates to a method of obtaining from a bacterial host a mature protein or polypeptide and thereby avoiding the need to treat the protein or polypeptide further to remove the signal sequence of other chemical substituents, such as an f-met (i.e., the formaldehyde group on its first methionine group), which are present on its precursor, as synthesized by the bacterial host.
It is known from Gilbert et al U.S. patent application, Ser. No. 913,533, filed June 8, 1978, and Villa-Komaroff et al, P.N.A.S. 75, 3727-3731 (1978) that a selected protein or polypeptide can be synthesized within a bacterial host and excreted through the membrane of the host by:
cleaving a cloning vehicle within its bacterial gene which codes for an extracellular or periplasmic carrier protein or polypeptide; PA1 forming a hybrid gene by inserting into the cleavage site a non-bacterial DNA fragment which codes for the selected protein or polypeptide; PA1 transforming the host with the cloning vehicle; and then PA1 culturing the transformed host to secrete the selected protein or polypeptide. PA1 (a) cleaving a cloning vehicle to form a cleavage site after a promoter of either (1) a bacterial or phage gene within the cloning vehicle or (2) a DNA fragment of the bacterial or phage gene; PA1 (b) forming a hybrid gene by inserting into the cleavage site a non-bacterial DNA fragment which codes for a precursor of the selected protein or polypeptide, including the signal sequence of the selected protein or polypeptide; PA1 (c) transforming the host with the cloning vehicle; and then PA1 (d) culturing the transformed host to secrete the selected protein or polypeptide.
However, the selected proteins and polypeptides, made by this method, have been obtained as fused proteins or polypeptides--the selected protein or polypeptide being fused to the bacterial carrier protein or polypeptide. As a result, additional steps have been required to cleave the selected protein or polypeptide from the bacterial carrier protein or polypeptide in order to obtain the selected protein or polypeptide, free of the bacterial carrier protein or polypeptide.
One method proposed to eliminate the fused bacterial carrier protein or polypeptide is to clone the codon for an unusual amino acid directly in front of the non-bacterial DNA fragment for the selected protein or polypeptide and to subject the selected protein or polypeptide, as produced by a transformed host, to chemical cleavage of the unusual amino acid. Itakura, et al, Science 198, 1056--1063 (1977). However, besides requiring additional steps to obtain the selected protein or polypeptide, free of the unusual amino acid, this method cannot be used to produce a selected protein or polypeptide containing the unusual amino acid. This is because such a protein or polypeptide would be destroyed by the chemical cleavage of that unusual amino acid.
Another alternative method, that has been proposed, has involved trimming back the DNA of the bacterial gene, so that the non-bacterial DNA fragment is directly after the translational start signal (ATG) of the bacterial DNA. However, this method produces in bacterial hosts the selected protein or polypeptide with an f-met, requiring further steps to obtain just the selected mature protein or polypeptide.